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Analysis on in vitro growth and haracterisation of metabolic athways of Mycoplasma suis linked to Pathogenesis

Schreiner, S A. Analysis on in vitro growth and haracterisation of metabolic athways of Mycoplasma suis linked to Pathogenesis. 2011, University of Zurich, Faculty of Science.

Abstract

The haemotrophic bacterium Mycoplasma suis is the causative agent of infectious anaemia in pigs (IAP), a worldwide and economically significant disease. The acute disease is characterised by severe anaemia, high bacteraemia, fever and high-grade hypoglycaemia. Antibiotic treatment using oxytetracyline protects the pigs against death, but does not eliminate the bacterium from the host. Hence, chronic low-grade M. suis infections are predominant. Clinical signs of the chronic form are mild anaemia, growth retardation in piglets, decreased reproductive efficiency in sows, and increased susceptibility to other infections. The crucial barrier to systematic analyses of the biology of M. suis is that M. suis as well as all known haemotrophic mycoplasmas has not been cultivable in vitro so far. Thus, M. suis research still relies on the splenectomised pig model, and on animal-toanimal passages, a method connected with serious ethical concerns. In this Ph.D. thesis presented, I describe efforts to establish an in vitro cultivation system for M. suis as a model system for all haemotrophic mycoplasmas with the aim to replaceanimal experiments. I attempted M. suis propagation by inoculating different Mycoplasma media with anti-coagulated blood from experimentally infected and acutely diseased pigs, and by systematically diversifying proved culture techniques for mycoplasmas. Some cultivation approaches used led to a kind of maintenance of M. suis over 12 weeks in liquid M. suis cultures. Scanning electron microscopicanalysis of subcultured M. suis cells on agar plates provided evidence: for the first time, propagated M. suis cells outside the natural host i.e. the pig. These propagated M. suis were packed in dense microcolonies consisting of small irregular M. suis nanocells indicating a kind of nanotransformation. Interestingly, these unique M. suis nanoforms are able to infect healthy porcine erythrocytes in vitro, thus indicating viability and infectivity. One main clinical sign of acute IAP is severe and often life-threatening hypoglycaemia caused by glucose consumption of M. suis itself. Due to the not-yetestablishedin vitro culture system and to missing whole genome sequences, little was known about the metabolism and the transport capacities of M. suis. Therefore, the second part of the Ph.D. thesis presented aimed to explore this part of the M. suis biology in more detail. For this, a random shotgun screening approach in combination with subsequent Southern blot hybridisation of genomic M. suis DNA was used.Thereby the transport system for the glucose uptake (i.e. the phosphoenolpyruvate:sugar phosphotransferase system PTSglc uptake system), the pre-protein secretion machinery (Sec-translocase), the phosphate (Pi)-specific import system (Pst-transporter) and several glycolytic enzymes of M. suis were identified and their genomic context was characterised. The genomic organisation of most of the genes identified was analogous to other mycoplasmas. Only the organisation of the Pst-transporter differs from other mycoplasmas. In M. suis, PhoU, the putative negative regulator of the Pho regulon, is not encoded together with the subunits of the Pst-transporter, whereas in other mycoplasmas, the pst-genes are organised in a polycistronic pstSCAB-phoU operon. Adhesion to erythrocytes is one of the main characteristics of haemotrophic mycoplasmas. Electron microscopic investigations revealed that the adhesion ismediated by fine bacterial fibrils. Recent studies have shown that a glycolytic enzyme i.e. the GAPDH protein MSG1 is involved in the adhesion. Within the present Ph.D.study a second glycolytic protein i.e. α-enolase of M. suis was identified by the above shotgun screening, and further characterised as potential adhesion and virulence factor. The M. suis α-enolase was expressed in Escherichia coli and the resulting purified recombinant protein was used to raise an anti-M. suis α-enolase rabbit serum and to perform characterisation assays. Interestingly, despite the missing classical signal sequence for export, M. suis α-enolase is a membrane-localised and surfaceaccessible protein with immunogenic and adhesive properties. Further studies arenecessary to evaluate the role of M. suis α-enolase as a putative part of the M. suis adhesion complex.

Abstract

The haemotrophic bacterium Mycoplasma suis is the causative agent of infectious anaemia in pigs (IAP), a worldwide and economically significant disease. The acute disease is characterised by severe anaemia, high bacteraemia, fever and high-grade hypoglycaemia. Antibiotic treatment using oxytetracyline protects the pigs against death, but does not eliminate the bacterium from the host. Hence, chronic low-grade M. suis infections are predominant. Clinical signs of the chronic form are mild anaemia, growth retardation in piglets, decreased reproductive efficiency in sows, and increased susceptibility to other infections. The crucial barrier to systematic analyses of the biology of M. suis is that M. suis as well as all known haemotrophic mycoplasmas has not been cultivable in vitro so far. Thus, M. suis research still relies on the splenectomised pig model, and on animal-toanimal passages, a method connected with serious ethical concerns. In this Ph.D. thesis presented, I describe efforts to establish an in vitro cultivation system for M. suis as a model system for all haemotrophic mycoplasmas with the aim to replaceanimal experiments. I attempted M. suis propagation by inoculating different Mycoplasma media with anti-coagulated blood from experimentally infected and acutely diseased pigs, and by systematically diversifying proved culture techniques for mycoplasmas. Some cultivation approaches used led to a kind of maintenance of M. suis over 12 weeks in liquid M. suis cultures. Scanning electron microscopicanalysis of subcultured M. suis cells on agar plates provided evidence: for the first time, propagated M. suis cells outside the natural host i.e. the pig. These propagated M. suis were packed in dense microcolonies consisting of small irregular M. suis nanocells indicating a kind of nanotransformation. Interestingly, these unique M. suis nanoforms are able to infect healthy porcine erythrocytes in vitro, thus indicating viability and infectivity. One main clinical sign of acute IAP is severe and often life-threatening hypoglycaemia caused by glucose consumption of M. suis itself. Due to the not-yetestablishedin vitro culture system and to missing whole genome sequences, little was known about the metabolism and the transport capacities of M. suis. Therefore, the second part of the Ph.D. thesis presented aimed to explore this part of the M. suis biology in more detail. For this, a random shotgun screening approach in combination with subsequent Southern blot hybridisation of genomic M. suis DNA was used.Thereby the transport system for the glucose uptake (i.e. the phosphoenolpyruvate:sugar phosphotransferase system PTSglc uptake system), the pre-protein secretion machinery (Sec-translocase), the phosphate (Pi)-specific import system (Pst-transporter) and several glycolytic enzymes of M. suis were identified and their genomic context was characterised. The genomic organisation of most of the genes identified was analogous to other mycoplasmas. Only the organisation of the Pst-transporter differs from other mycoplasmas. In M. suis, PhoU, the putative negative regulator of the Pho regulon, is not encoded together with the subunits of the Pst-transporter, whereas in other mycoplasmas, the pst-genes are organised in a polycistronic pstSCAB-phoU operon. Adhesion to erythrocytes is one of the main characteristics of haemotrophic mycoplasmas. Electron microscopic investigations revealed that the adhesion ismediated by fine bacterial fibrils. Recent studies have shown that a glycolytic enzyme i.e. the GAPDH protein MSG1 is involved in the adhesion. Within the present Ph.D.study a second glycolytic protein i.e. α-enolase of M. suis was identified by the above shotgun screening, and further characterised as potential adhesion and virulence factor. The M. suis α-enolase was expressed in Escherichia coli and the resulting purified recombinant protein was used to raise an anti-M. suis α-enolase rabbit serum and to perform characterisation assays. Interestingly, despite the missing classical signal sequence for export, M. suis α-enolase is a membrane-localised and surfaceaccessible protein with immunogenic and adhesive properties. Further studies arenecessary to evaluate the role of M. suis α-enolase as a putative part of the M. suis adhesion complex.

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